Although the cardiovascular morbidity and mortality induced by cigarette smoking exceeds that attributable to lung cancer, the molecular basis of smoking induced tissue injury remains unclear. Considerable evidence supports a role for oxidant stress, and we have previously shown a dose response relationship between smoking and urinary isoprostane (iP) excretion, a reduction in iP excretion on quitting and suppression of elevated iPs in smokers with replacement of depleted vitamin C. Endothelial function is impaired in smokers, a harbinger of overt atherosclerosis and this defect is corrected by vitamin C supplementation. However, little is known of the effects of antioxidant vitamins on DNA and protein oxidation and we have some evidence that vitamin C may exacerbate DNA adduct formation. Interest has now developed in long term nicotine supplementation for smokers who cannot or will not quit. However, little is known of the chronic cardiovascular pharmacology of nicotine and several studies have shown impaired EC function in smokers switching to nicotine. The present studies are designed to investigate comprehensively the impact of nicotine on oxidative stress and its effect on EC function in humans, using novel approaches to the coordinate assessment of lipid peroxidation, protein oxidation and DNA modification in vivo. They will be complemented by studies of the impact of nicotine on oxidative stress and atherogenesis in mouse models employing novel approaches to proteomic analysis of the vasculature and circulating cells. Specific Aim 1: To assess the dose related impact of cigarette smoking on novel indices of lipid peroxidation, protein oxidation and DNA modification and on endothelial function in vivo. To assess the impact of vitamin C supplementation on these indices in cigarette smokers. To assess the impact of vitamin C supplementation on the disordered neutrophil protein expression found in smokers. Specific Aim 2: To assess the dose related impact of nicotine substitution on indices of oxidant stress, endothelial function and smoking induced neutrophil protein expression in smokers switched to nicotine. Specific Aim 3: To define the impact of chronic exposure to cigarette smoke on atherogenesis in the apobec-l/ LDLR double knockout (dko) mouse. To assess the effect of nicotine substitution on established smoking related atherosclerosis with and without continued smoke exposure and to assess the effect of nicotine de novo on atherogenesis in this model. Vascular tissues will be harvested to provide specimens for analysis of modified hyaluronan, fibrinogen and LDL in other projects. Proteomic analysis in the vasculature will assess the impact of disturbed flow and seek patterns analogous to those evident in human neutrophils in Specific Aim 1.